EP1204608B9 - Method of purifying water, suitable bacteria for the method and use thereof - Google Patents

Method of purifying water, suitable bacteria for the method and use thereof Download PDF

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EP1204608B9
EP1204608B9 EP00948037A EP00948037A EP1204608B9 EP 1204608 B9 EP1204608 B9 EP 1204608B9 EP 00948037 A EP00948037 A EP 00948037A EP 00948037 A EP00948037 A EP 00948037A EP 1204608 B9 EP1204608 B9 EP 1204608B9
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deposit number
number dsm
water
bioreactor
waste water
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German (de)
French (fr)
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EP1204608A2 (en
EP1204608B1 (en
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Jussi Uotila
Gennadi Zaitsev
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TEKNO-FOREST OY
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Tekno-Forest Oy
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • the invention relates to a method of purifying waste water biologically, and to bacteria and a mixed bacterial population suitable for the method and the use thereof.
  • the invention further relates to a bioreactor comprising said bacteria or mixed population.
  • water can be purified both by physical and chemical means, for example by sedimentation, filtration or flocculation (WO94/5866 and WO88/5334).
  • biological purification wherein the water to be purified is brought into contact with microorganisms that decompose pollution agents.
  • Biological water treatment methods are suited for use both in conventional water treatment plants and industrial waste water treatment plants.
  • Biological water treatment has also been tested in systems where water is recycled (FI 964141).
  • Biological water treatment is also needed to purify seep water of a dump, for example, before the seep water is discharged into the environment.
  • the biological purifying method is, however, more difficult to control than the physical or chemical purifying methods. Firstly, microorganisms to decompose pollution agents must be found. Secondly, the microorganisms must be capable of easily surviving and reproducing under conditions during the water treatment process. In other words, the microorganisms used for purifying water must be competitive ones so as to prevent other organisms in the water from overruling. In addition, the microorganisms used for purifying water must not be sensitive to the changes in their environment that often occur during water treatment processes when the load varies.
  • microorganisms have been used for purifying water, including bacteria and protozoa, such as the ciliates.
  • Bacteria that have often been used include species of the Pseudomas genus, but also members of the Alcagenes, Acinetobacter or Rhodococcus genera are often used. Mixed populations, some identified and some unidentified, comprising a great number of different microorganisms are often used. Aerobic or facultative microorganisms are best suited to purifying water, in which case it is appropriate to pump air into the water to be purified so as to make the purification process more efficient.
  • US-A-5 679 568 discloses decomposition of a halogenated organic acid and/or of aliphatic organochlorine compounds by certain organisms including Pseudomonas and Xanthobacter and particularly a new Renobacter strain.
  • EP-A-915 061 relates to how to remove the detrimental effect of detergents on microorganism growth in order to improve e.g. industrial fermentation processes.
  • the detergent's detrimental effect is removed by adding hydrolysing enzymes to the liquid i.e. the removal of the detergent from the wastewater is enzymatic not biological.
  • US-A-4 317 885 relates to the use of a particular strain of Pseudomonas fluorescens in removing detergents and other pollutants from wastewater.
  • the strain used is an obligate aerobe, which means that it is not capable of denitrification, which is essential in removing nitrogenous compounds from wastewater.
  • the growth medium When microorganisms, are cultivated, the growth medium should normally be sterilized so as to prevent the cultivation from becoming contaminated by external organisms. Since large amounts of water are processed while purifying waste water, the amount of necessary biomass for the biological purification is also large. To produce such biomass under sterile conditions is both laborious and expensive; hence, it would be most desirable if the biomass could be produced under non-sterile conditions without any danger of becoming contaminated.
  • the present invention now provides a novel fermentation technology with no need to sterilize. This is possible when microorganisms particularly suitable for the method are used and these microorganisms are fed on nutrients suitable for them.
  • the present invention relates to microorganisms that are surprisingly well suited to biological purification of waste water. These microorganisms meet particularly well the aforementioned requirements set for microorganisms suitable for the biological purification of water.
  • the microorganisms of the invention are so specific that their biomass can be produced under non-sterile conditions by using a growth medium where other microorganisms are unable to compete. This enables large savings in the costs and energy consumption of a biological water purification process, the purification results also being excellent. Water purified according to the invention is even recyclable.
  • the invention thus relates to the bacteria Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas sp. DT-2, subsequently identified as Pseudomonas azelaica having the deposit number DSM 12561, and the former Pseudomonas sp. now being Rhizobium sp. and having the deposit number DSM 12562. Later 16S rDNA analyses have shown that this bacterium most closely resembles the members of the Rhizobium genus, so hereafter, it will be considered as one of them.
  • the invention further relates to the following bacterial strains promoting water purification: Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and Xanthobacter sp. DT-13 having the deposit number DSM 13519.
  • DSM 12560 - 12562 have been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on 1 December 1998, and DSM 13516 - 13519 on 29 May 2000.
  • the invention further relates to a bacterial mixed population characterized by comprising the bacterium Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and/or Rhizobium sp. DT-5 having the deposit number DSM 12562.
  • the invention further relates to the use of the bacterial mixed population in waste water treatment and to a method of purifying waste water, characterized by purifying water biologically by a mixed population comprising the microorganisms Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and Rhizobium sp. DT-5 having the deposit number DSM 12562.
  • the invention further relates to a bioreactor characterized by comprising the microorganisms Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and Rhizobium sp. DT-5 having the deposit number DSM 12562.
  • a bioreactor is a reactor in which a biological purification process is conducted.
  • Microorganisms growing in a soap mixture were enriched from waste water of an industrial plant and then adapted by cultivating them in a bioreactor comprising waste water from a dump.
  • Three bacterial strains were thus isolated that were superior to the others.
  • Said bacterial strains are Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561 and Rhizobium sp. DT-5 having the deposit number DSM 12562.
  • These bacteria can be cultivated in tap water comprising about 1 - 4 g/l of soap.
  • the isolated bacterial strains can particularly decompose compounds that do not decompose easily, such as chlorophenoles, polycyclic aromatic hydrocarbons (PAH compounds) and oils. They also remove heavy metals.
  • Bacillus sp. DT-1, Pseudomonas azelaica DT-2 and Rhizobium sp. DT-5 further tend to flocculate, in which case they form a so-called bionetwork, which comprises lumps comprising microorganisms and other particles and which promotes the purification.
  • waste water treatment results are achieved when biological water purification utilizes a bacterial mixed population comprising the bacteria Bacillus sp. DT-1, Pseudomonas azelaica DT-2 and Rhizobium sp. DT-5.
  • the bacterial mixed population may further comprise other microorganism strains that are useful in water treatment and that have a favourable combined effect on the purification capacity.
  • microorganism strains DT-1, DT-2, and/or DT-5 are used together with one or more bacterial strains from the group Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and Xanthobacter sp. DT-13 having the deposit number DSM 13519.
  • Said four strains were isolated from the biofilm of the last unit of a four cascade bioreactor for treating water containing a mixture of soaps.
  • DT-6, DT-10, DT-12 and DT-13 improve the immobilization properties of the biofilm to supporting matrices when they are mixed with strains DT-1, DT-2 and DT-5. Association of the strains also improves the treatment process of waste water as a result of more tolerance of the biofilm formed against poisonous substances.
  • Bacillus sp. DT-1 is a rod which is about 1.0 - 1.2 ⁇ m in width and 3.0 - 6.0 ⁇ m in length. Partial sequencing of the 16S rDNA shows a similarity of 99.3% to B. cereus and 100% to B. thuringiensis.
  • Pseudomonas azelaica DT-2 is a rod which is 0.5 - 0.7 ⁇ m in width and 1.5 - 3.0 ⁇ m in length with 1 - 3 polar flagella and lacking fluorescent pigments.
  • the partial sequencing of the 16S rDNA is 99.8% similar to Ps. azelaica . It reacts as follows: Lysis by 3% KOH + Aminopeptidase (Cerny) + Lecithinase - Utilization of arabinose - adipat + mannitol - gluconat + caprat +
  • Rhizobium sp. DT-5 is a rod which is 0.5 - 0.7 ⁇ m in width and 1.5 - 3.0 ⁇ m in length. Partial 16S rDNA sequencing shows a 98.6% similarity to R. giardinii and 98.6% similarity to Phyllobacterium myrisinacearum. Physiological test results are given below. They do not confirm any of these genera. Lysis by 3% KOH + Aminopeptidase (Cerny) + Anaerobic growth - Simmons citrate + Utilization of arabinose + mannose + mannitol + adipat -
  • the retention time (in minutes) is shown on the x-axis of Figures 2a and 3a, and the intensity of a peak is shown on the y-axis of the same figures.
  • the corresponding prints of the fatty acid analyses are shown in Figures 2b, 3b and 4.
  • the profile of the fatty acids of DT-1 is typical of the B. cereus group.
  • the profile of DT-2 is typical of the RNA group I of the pseudomonads, and the profile of DT-5 points to the Rhizobium group.
  • Pseudomonas azelaica DT-6 is a 0.5 - 0.7 ⁇ m wide and 1.5 - 3.0 ⁇ m long gram-negative motile rod having 1 - 3 polar flagella and lacking fluorescent pigments. Its fatty acid analysis print ( Figure 5) is typical of the RNA group I of the pseudomonads. The partial sequencing of the 16S rDNA shows a 99.8% similarity to Ps. azelaica.
  • DT-6 has the following physiological reactions: Lysis by 3% KOH + Aminopeptidase (Cerny) + Oxidase + Catalase + ADH + NO 2 from NO 3 + Denitrification weak Urease - Hydrolysis of gelatin - Lecithinase - Utilization of (API 20NE) glucose + arabinose - adipat + malat + mannitol - gluconat + caprat +
  • Azospirillum sp. DT-10 is a 0.8 - 1.2 ⁇ m wide and 2.0 - 4.0 ⁇ m long gram-negative rod. Its fatty acid analyisis print ( Figure 6) is typical of the ⁇ -subgroup of the proteobacteria and points to the genus Azospirillum.
  • the partial sequencing of the 16S rDNA shows similarities between 92% and 97.4% to different members of the genus Azospirillum. The highest similarity 97.4% was found to Azospirillum lipoferum.
  • the physiological reactions of DT-10 are shown below. They point to the genus Azospirillum but are not typical of A. lipoferum.
  • DT-10 is possibly a new species of this genus. Lysis by 3% KOH weak Aminopeptidase (Cerny) + Oxidase + Catalase + NO 2 from NO 3 + Urease + ADH - Hydrolysis of gelatin - esculin - Utilization of (sole carbon source) glucose - arabinose - adipat - malat + mannitol - phelyacetat - citrate - caprat - gluconat - maltose - n-acetylglucosamin - ⁇ -ketoglutarate + sucrose - m-inositol - D-fructose + rhamnose - arabitol - ribose - Growth at 41 °C - with 3% NaCl -
  • Ancylobacter aquaticus DT-12 is a gram-negative curved rod which is 0.5 - 0.7 ⁇ m in width and 1.5 - 2.0 ⁇ m in length.
  • the partial sequence of the 16S rDNA shows a similarity of 98.8% to Ancylobacter aquaticus.
  • Thiobacillus novellus shows a similarity of 97.8%.
  • the fatty acids ( Figure 7) point to the ⁇ -proteobacteria. The physiological tests as shown below clearly identify the species Ancylobacter aquaticus.
  • Xanthobacter sp. DT-13 is an irregular, motile, gram-negative rod which is 0.8 - 1.0 ⁇ m in width and 1.5 - 3.0 ⁇ m in length.
  • the partial sequences of the 16S rDNA show similarities of 98.5% to 99.3% to different members of the genus Xanthobacter.
  • X. falvus shows the highest similarity (99.3%).
  • the profile of the fatty acids is typical of the subclass of ⁇ -proteobacteria. The physiological tests are not able to distinguish reliably between the species of this genus (i.e. no pigment production detected, no slime production, etc.).
  • the above-described bacteria are suited for use in purifying waste water.
  • the bacteria can then be first grown in a minimal salt medium (KSN) in a shaker. Soy pepton (0.5 g/l), trypton (0.1 g/l), glucose (0.2 g/l) and potassium acetate (0.3 g/l) may be added, if desired.
  • KSN minimal salt medium
  • Soy pepton (0.5 g/l), trypton (0.1 g/l), glucose (0.2 g/l) and potassium acetate (0.3 g/l) may be added, if desired.
  • the growing temperature of the bacteria is about 20 - 30°C.
  • the volume of the culture is then increased in order to produce the necessary biomass for purifying the water. This stage no longer needs to be conducted under sterile conditions, in which case tap water wherein about 0.5 - 4 g/l of soap has been added can be used as the growth medium.
  • the soap used is preferably a mixture containing anionic, cationic, amphoteric and non-ionic tensides. It is preferable to use a mixture of different soaps, such as cleaning agents, fabric conditioners and detergents for clothes and dishes.
  • the bacteria are grown as a submerged culture with air pumped thereto.
  • the biomass can be produced as a batch culture, but preferably, it is produced as a continuous culture, or chemostat culture. It is preferable to use a carrier in the production of the biomass. Any common carrier, for example a plastic one, is suitable for this purpose.
  • the produced biomass is then transferred into a water treatment reactor, into which the water to be purified is conveyed.
  • a carrier for the bacteria is also used in the reactor, the carrier preferably being the same as used in the production of the biomass.
  • the carrier is preferably one having a specific density lower than 1 g/cm 3 .
  • the carrier is generally held in place in a tank by means of a net ('fixed carrier'), for example, but sometimes the carrier is allowed to float freely in the tank ('swimming carrier').
  • the method of the invention is suited particularly to purifying seep water of a dump, which is here described in closer detail with reference to Figure 1.
  • a dump is usually surrounded by a ditch to collect the seep water.
  • Seep water refers to water seeping from a dump due to rain and ground water.
  • This seep water containing both surface water and cavity water is usually first conveyed to a tank wherefrom the water is conveyed through a purification process before being discharged into the environment.
  • the seep water obtained both from deep and shallow ground is preferably first conveyed to a settlement basin, from which the water is filtered through an inlet pipe 1 to a filtrate well 2, and from there, through a transfer pipe 8 to a bioreactor 3 containing said bacteria and a carrier 5.
  • the bacteria form a so-called biofilm around the carrier.
  • the carrier with its bacteria is usually kept below the surface of the water by means of a net.
  • the bioreactor preferably comprises one or more separating walls 6 arranged to force the water to circulate in the reactor.
  • the separating walls may be arranged on opposite walls, for example, as shown in Figure 1.
  • the reactor usually further comprises an aerator 9 for conveying air into the reactor through an aeration pipe 4.
  • the bioreactor further comprises an outlet pipe 7, through which processed water is discharged from the reactor.
  • grey water refers to waste water other than that originating from lavatories, e.g. water from showers, handbasins, bath tubs and laundry rooms.
  • the purification method of the invention is also suited to purifying waste water from lavatories, which is called black water.
  • the method of the invention can also be used to purify laundry and industrial waste water, which often contains a large amount of organic waste, such as oil, polycyclic aromatic hydrocarbons (PAH compounds) and/or heavy metals.
  • PAH compounds polycyclic aromatic hydrocarbons
  • the method is also suitable for purifying waste water originating from food industry and water in swimming pools.
  • KSN sterilized minimum salt medium
  • Bacillus sp. DT-1, Pseudomonas azelaica DT-2 and Rhizobium sp. DT-5 were each transferred to 200 ml of sterilized minimum salt medium (KSN) of the following composition (g/l of distilled water): K 2 HPO 4 ⁇ 3H 2 O - 1.0, NaH 2 PO 4 ⁇ 2H 2 O - 0.25, (NH 4 ) 2 SO 4 - 0.1, MgSO 4 ⁇ 7H 2 O - 0.04, Ca(NO 3 ) 2 ⁇ 4H 2 O - 0.01, yeast extract - 0.05, pH 7.0 - 7.3, and soap mixture about 1 g/l.
  • KSN sterilized minimum salt medium
  • the soap mixture contained about equal amounts of the following detergents: laundry soap, Comfort, Cleani Family -fabric conditioner, Cleani Color, Serto Ultra, Bio Luvil, Ariel Futur, Omo Color, Tend Color, Tend Mega, Tend Total and Eko Kompakt (about 1g/l in total).
  • the bacteria were grown in a shaker (150 - 200 rpm), at 28°C.
  • the fermenter contained unsterilized tap water and a total of 4 g/l of the aforementioned soap mixture, and a plastic carrier containing polyethene and having a specific density of about 0.8 g/cm 3 .
  • the carrier was kept below the surface of the liquid by means of a net.
  • the cultivation now continued under non-sterile conditions to a turbidity of about 2 (600 nm), and then as a chemostat culture.
  • a first inoculum obtained from the fermenter was then introduced into a bioreactor (6 m 3 ) according to Figure 1, diluted 1:10.
  • the bioreactor contained seep water from a municipal dump which was first collected into a tank, wherefrom it was then transferred to a settlement basin for removal of solid matter and next, to a filtrate well, wherefrom it was pumped to the bioreactor. In principle, the system works by gravity, the only necessary pump being a submersible pump in the filtrate well.
  • the bioreactor contained the same carrier as the fermenter used for producing the biomass. The carrier was kept below the liquid level by means of a net. The bacteria flocculated at the end of the bioreactor. The purification process was continuous, operating at a capacity of about 100 m 3 /24 hours. Air was pumped so as to keep the oxygen content of the water to be processed > 7 mg/l.
  • a bioreactor arranged according to Example 1 was used for purification of seep water from a municipal dump.
  • the average COD of the waste water to be purified was about 800 mg - 6 g O 2 /l.
  • the waste water contained chlorophenoles, PAH compounds and oil, for example. The removal of these subsctances from the waste water was monitored. According to Nordtest's technical report no. 329 (accepted 9603), the compounds were defined by a gas chromatograph equipped with a mass-selective detector. The results are shown in Table 2.
  • Waste water from a municipal waste water plant was purified both in a manner conventionally used in the plant and by the method of the invention.
  • waste water was purified by first conveying the waste water into a preliminary settlement basin in order to precipitate the solids onto the bottom.
  • the preliminary settled water was then conveyed to an aerobic treatment basin, whereto ferrous sulphate for precipitating phosphate, and polyamine for precipitating biosludge were added.
  • ferrous sulphate for precipitating phosphate, and polyamine for precipitating biosludge were added.
  • the water was further conveyed to a secondary settlement basin.
  • the purification system of the invention comprised five tanks whose total volume was 7.5 m 3 , the tanks being interconnected in the following order: two anaerobic tanks, whereto bacteria DT-1, DT-2 and DT-5 were added without a carrier, one aerobic tank whereto a carrier was attached (by means of a net) on which the bacteria DT-1, DT-2 and DT-5 were immobilized, and two sedimentation tanks.
  • the temperature was 8 - 15°C.
  • the flow rate was 7.5 m 3 /24 hours of waste water.
  • the aeration was conducted by recycling the water through the carrier. The results are shown in Table 3.
  • the purification results achieved by the method of the invention were either as good as or better than those achieved by the conventional method, and energy consumption was significantly lower.
  • the energy consumption in treating one cubic metre of water was 0.23 kWh at the municipal waste water treatment plant, and 0.05 - 0.1 kWh when the method of the invention was used.
  • the system comprised five tanks whose total volume was 6.5 m 3 , the tanks being interconnected in the following order: two anaerobic tanks without a carrier into which the DT-1, DT-2 and DT-5 were added, one aerobic tank whereto a carrier was attached on which the bacteria DT-1, DT-2 and DT-5 were immobilized, and two sedimentation tanks.
  • the temperature was 8 - 15°C.
  • the flow rate was 0.5 - 5 m 3 of waste water per 24 hours.
  • the aeration was conducted by recycling the water through the carrier.
  • the energy consumption was 0.05 - 0.5 kWh.
  • Table 4 The results are shown in Table 4.
  • Waste water from a coating metal industry plant was purified by a system whose effective treatment part comprised six anaerobic and twelve aerobic tanks.
  • the bacteria DT-1, DT-2 and DT-5 which were immobilized on a carrier attached by nets, were added to all anaerobic and aerobic tanks. Each tank held 2 l.
  • the entire system comprised 23 tanks whose total volume was 70 l, the tanks being interconnected in the following order: six anaerobic tanks (effective treatment volume), one sedimentation tank, six aerobic tanks (effective treatment volume), one sedimentation tank, six aerobic tanks (effective treatment volume), and two tanks for calcium chloride and sodium hydroxide treatments to precipitate the biomass and heavy metals. Before the treatment, the original waste water was diluted five times by gray water.
  • mineral salts were added as follows: NH + / 4 2 - 10 mg/l, NO - / 3 5 - 20 mg/l. Mg 2+ 2 - 10 mg/l, Ca 2+ 0.5 - 2 mg/l, SO 4 2- 1 - 10 mg/l and PO 4 3- 2 - 20. mg/l. The temperature was 20 - 35°C and the flow rate 12 l of water per 24 hours. The results are shown in Table 5.
  • the effective part of the system comprised three aerobic tanks whose single volume was 0.2 m 3 .
  • the entire system comprised six tanks whose total volume was 2.8 m 3 , the tanks being interconnected in the following order: one tank for collecting grey water, three aerobic tanks comprising a fixed carrier on which the bacteria DT-1, DT-2 and DT-5 were immobilized (effective treatment volume), one aerobic tank without a carrier and one sedimentation tank, and, subsequently, a filtering system and a UV-light treatment system.
  • the temperature was 20 - 35°C.
  • the flow rate was about 1 m 3 per 24 hours. The results are shown in Table 6.
  • the effective treatment part of the system comprised two aerobic tanks having the volume of 1 m 3 , the tanks comprising a swimming carrier on which DT-1, DT-2 and DT-5 were immobilized.
  • the entire system comprised ten tanks whose total volume was 23 m 3 , the tanks being interconnected in the following order: one tank for collecting grey water, two aerobic tanks comprising a swimming carrier (effective treatment volume), one sedimentation tank, three aerobic tanks comprising a fixed carrier with its bacteria (effective treatment volume), one aerobic tank without a carrier, and two sedimentation tanks.
  • the temperature of the water was 20 - 35°C, the flow rate 1 m 3 of waste water per 24 hours.
  • the results are shown in Table 7. Parameter Before treatment After treatment COD cr mg O 2 /l 200 - 450 25 - 35 Total phosphorus mg P/l 1 - 2 ⁇ 0.1 pH 8.5-9 7-8
  • Biomass of strains DT-1, DT-2, DT-5, DT-6, DT-10, DT-12 and DT-13 was produced and immobilzed on a carrier as set forth in Example 1, and the amount of biomass on the carrier was weighed.
  • the weight of one disc of the carrier was 72 ⁇ 1 g.
  • the weight of one disc of the carrier was 119 ⁇ 13, i.e. the wet weight of the biomass was 47 ⁇ 11 g per disc.
  • the weight of one disc of carrier was 172 ⁇ 16, i.e. the wet weight of the biomass was 91 ⁇ 16.
  • the results show that DT-6, DT-10, DT-12 and DT-13 increased the immobilized biomass about twofold.

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Abstract

A method of purifying waste water biologically by using three particularly suitable bacteria: Bacillus sp. DT-1, Pseudomonas azelaica, DT-2, and/or Rhizobus sp. DT-5, or mixed populations thereof. The invention further relates to the bacteria and the mixed populations and use thereof in purifying waste water. The invention further relates to a bioreactor including the bacteria.

Description

FIELD OF THE INVENTION
The invention relates to a method of purifying waste water biologically, and to bacteria and a mixed bacterial population suitable for the method and the use thereof. The invention further relates to a bioreactor comprising said bacteria or mixed population.
BACKGROUND OF THE INVENTION
Conventionally, water can be purified both by physical and chemical means, for example by sedimentation, filtration or flocculation (WO94/5866 and WO88/5334). In order to remove organic compounds and other compounds that are difficult to purify it is also preferable to use so-called biological purification wherein the water to be purified is brought into contact with microorganisms that decompose pollution agents. Biological water treatment methods are suited for use both in conventional water treatment plants and industrial waste water treatment plants. Biological water treatment has also been tested in systems where water is recycled (FI 964141). Biological water treatment is also needed to purify seep water of a dump, for example, before the seep water is discharged into the environment.
The biological purifying method is, however, more difficult to control than the physical or chemical purifying methods. Firstly, microorganisms to decompose pollution agents must be found. Secondly, the microorganisms must be capable of easily surviving and reproducing under conditions during the water treatment process. In other words, the microorganisms used for purifying water must be competitive ones so as to prevent other organisms in the water from overruling. In addition, the microorganisms used for purifying water must not be sensitive to the changes in their environment that often occur during water treatment processes when the load varies.
Many kinds of microorganisms have been used for purifying water, including bacteria and protozoa, such as the ciliates. Bacteria that have often been used include species of the Pseudomas genus, but also members of the Alcagenes, Acinetobacter or Rhodococcus genera are often used. Mixed populations, some identified and some unidentified, comprising a great number of different microorganisms are often used. Aerobic or facultative microorganisms are best suited to purifying water, in which case it is appropriate to pump air into the water to be purified so as to make the purification process more efficient.
US-A-5 679 568 discloses decomposition of a halogenated organic acid and/or of aliphatic organochlorine compounds by certain organisms including Pseudomonas and Xanthobacter and particularly a new Renobacter strain.
EP-A-915 061 relates to how to remove the detrimental effect of detergents on microorganism growth in order to improve e.g. industrial fermentation processes. The detergent's detrimental effect is removed by adding hydrolysing enzymes to the liquid i.e. the removal of the detergent from the wastewater is enzymatic not biological.
US-A-4 317 885 relates to the use of a particular strain of Pseudomonas fluorescens in removing detergents and other pollutants from wastewater. The strain used is an obligate aerobe, which means that it is not capable of denitrification, which is essential in removing nitrogenous compounds from wastewater.
When microorganisms, are cultivated, the growth medium should normally be sterilized so as to prevent the cultivation from becoming contaminated by external organisms. Since large amounts of water are processed while purifying waste water, the amount of necessary biomass for the biological purification is also large. To produce such biomass under sterile conditions is both laborious and expensive; hence, it would be most desirable if the biomass could be produced under non-sterile conditions without any danger of becoming contaminated. The present invention now provides a novel fermentation technology with no need to sterilize. This is possible when microorganisms particularly suitable for the method are used and these microorganisms are fed on nutrients suitable for them.
SUMMARY OF THE INVENTION
The present invention relates to microorganisms that are surprisingly well suited to biological purification of waste water. These microorganisms meet particularly well the aforementioned requirements set for microorganisms suitable for the biological purification of water. In addition, the microorganisms of the invention are so specific that their biomass can be produced under non-sterile conditions by using a growth medium where other microorganisms are unable to compete. This enables large savings in the costs and energy consumption of a biological water purification process, the purification results also being excellent. Water purified according to the invention is even recyclable.
The invention thus relates to the bacteria Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas sp. DT-2, subsequently identified as Pseudomonas azelaica having the deposit number DSM 12561, and the former Pseudomonas sp. now being Rhizobium sp. and having the deposit number DSM 12562. Later 16S rDNA analyses have shown that this bacterium most closely resembles the members of the Rhizobium genus, so hereafter, it will be considered as one of them. The invention further relates to the following bacterial strains promoting water purification: Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and Xanthobacter sp. DT-13 having the deposit number DSM 13519. DSM 12560 - 12562 have been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on 1 December 1998, and DSM 13516 - 13519 on 29 May 2000.
The invention further relates to a bacterial mixed population characterized by comprising the bacterium Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and/or Rhizobium sp. DT-5 having the deposit number DSM 12562.
The invention further relates to the use of the bacterial mixed population in waste water treatment and to a method of purifying waste water, characterized by purifying water biologically by a mixed population comprising the microorganisms Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and Rhizobium sp. DT-5 having the deposit number DSM 12562.
The invention further relates to a bioreactor characterized by comprising the microorganisms Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and Rhizobium sp. DT-5 having the deposit number DSM 12562. A bioreactor is a reactor in which a biological purification process is conducted.
DRAWINGS
  • Figure 1 schematically shows a purification system for seep water,
  • Figure 2a shows a profile of the fatty acids of bacterial strain DT-1,
  • Figure 2b is a print of a fatty acid analysis of bacterial strain DT-1,
  • Figure 3a shows a profile of the fatty acids of bacterial strain DT-2,
  • Figure 3b is a print of a fatty acid analysis of bacterial strain DT-2,
  • Figure 4 is a print of a fatty acid analysis of bacterial strain DT-5,
  • Figure 5 is a print of a fatty acid analysis of bacterial strain DT-6,
  • Figure 6 is a print of a fatty acid analysis of bacterial strain DT-10,
  • Figure 7 is a print of a fatty acid analysis of bacterial strain DT-12, and
  • Figure 8 is a print of a fatty acid analysis of bacterial strain DT-13.
    • DETAILED DESCRIPTION OF THE INVENTION
    Microorganisms growing in a soap mixture were enriched from waste water of an industrial plant and then adapted by cultivating them in a bioreactor comprising waste water from a dump. Three bacterial strains were thus isolated that were superior to the others. Said bacterial strains are Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561 and Rhizobium sp. DT-5 having the deposit number DSM 12562. These bacteria can be cultivated in tap water comprising about 1 - 4 g/l of soap. Extremely few microorganisms can actively grow under such conditions; therefore, this growth medium needs not be sterilized when biomass of said three bacteria is being produced. The strains tolerate as high amounts of soap as about 40 g/l. They grow best in a soap content of about 0.3 - 0.5 g/l.
    In addition to being capable of growing in a growth medium where most other bacteria are incapable of reproducing, said bacterial strains are extremely efficient in removing the organic load of waste water. This is usually measured as total COD, which means the total chemical oxygen consumption (mg O2/l). The isolated bacterial strains can particularly decompose compounds that do not decompose easily, such as chlorophenoles, polycyclic aromatic hydrocarbons (PAH compounds) and oils. They also remove heavy metals.
    The bacteria Bacillus sp. DT-1, Pseudomonas azelaica DT-2 and Rhizobium sp. DT-5 further tend to flocculate, in which case they form a so-called bionetwork, which comprises lumps comprising microorganisms and other particles and which promotes the purification.
    Particularly good waste water treatment results are achieved when biological water purification utilizes a bacterial mixed population comprising the bacteria Bacillus sp. DT-1, Pseudomonas azelaica DT-2 and Rhizobium sp. DT-5. In addition to these three strains, the bacterial mixed population may further comprise other microorganism strains that are useful in water treatment and that have a favourable combined effect on the purification capacity.
    The best purification results are achieved when the microorganism strains DT-1, DT-2, and/or DT-5 are used together with one or more bacterial strains from the group Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and Xanthobacter sp. DT-13 having the deposit number DSM 13519. Said four strains were isolated from the biofilm of the last unit of a four cascade bioreactor for treating water containing a mixture of soaps. They can be grown in the same growth medium and under the same conditions as DT-1, DT-2 and DT-5. DT-6, DT-10, DT-12 and DT-13 improve the immobilization properties of the biofilm to supporting matrices when they are mixed with strains DT-1, DT-2 and DT-5. Association of the strains also improves the treatment process of waste water as a result of more tolerance of the biofilm formed against poisonous substances.
    Bacillus sp. DT-1 is a rod which is about 1.0 - 1.2 µm in width and 3.0 - 6.0 µm in length. Partial sequencing of the 16S rDNA shows a similarity of 99.3% to B. cereus and 100% to B. thuringiensis. In identification tests DT-1 reacted as indicated below:
    Anaerobic growth +
    VP reaction +
    pH in VP broth 4.8
    Growth in medium pH 5.7 +
    2% NaCl +
    5% +
    7% -
    10% -
    Lysozyme broth +
    Acid from
    L-arabinose -
    D-xylose -
    D-mannitol -
    D-fructose +
    Lecithinase +
       Hydrolysis of:
       casein +
       Tween 80 weak
    aesculin +
    Use of propionate -
    Indol reaction -
    Phenylalanine deaminase +
    Hemolysis +
    Growth in penicillin 900U/ml +
    Pseudomonas azelaica DT-2 is a rod which is 0.5 - 0.7 µm in width and 1.5 - 3.0 µm in length with 1 - 3 polar flagella and lacking fluorescent pigments. The partial sequencing of the 16S rDNA is 99.8% similar to Ps. azelaica. It reacts as follows:
    Lysis by 3% KOH +
    Aminopeptidase (Cerny) +
    Lecithinase -
    Utilization of arabinose -
    adipat +
    mannitol -
    gluconat +
    caprat +
    Rhizobium sp. DT-5 is a rod which is 0.5 - 0.7 µm in width and 1.5 - 3.0 µm in length. Partial 16S rDNA sequencing shows a 98.6% similarity to R. giardinii and 98.6% similarity to Phyllobacterium myrisinacearum. Physiological test results are given below. They do not confirm any of these genera.
    Lysis by 3% KOH +
    Aminopeptidase (Cerny) +
    Anaerobic growth -
    Simmons citrate +
    Utilization of arabinose +
    mannose +
    mannitol +
    adipat -
    Other morphological, physiological and biochemical characteristics of bacterial strains DT-1, DT-2 and DT-5 are shown in Table 1.
    Figure 00080001
    Figure 00090001
    Figure 00100001
    Furthermore, the profiles of the fatty acids of bacterial strains DT-1, DT-2 and DT-5 were determined and they are shown in Figures 2 to 4. The bacteria were grown 24 hours at 28°C on tryptic soy broth agar and methyl esters were prepared for the fatty acid analysis of the whole cell, as described in publication Structure and composition of biological slimes on paper and board machines. Appl. Environ, Microbiol. 60:641-653 by Väisänen, O.M., E-L. Nurmiaho-Lassila, S.A. Marmo and M.S. Salkinoja-Salonen (1994). An aerobic TSBA library, version 3.9 (MIDI Inc., Newark, DE, USA), was used. The retention time (in minutes) is shown on the x-axis of Figures 2a and 3a, and the intensity of a peak is shown on the y-axis of the same figures. The corresponding prints of the fatty acid analyses are shown in Figures 2b, 3b and 4. The profile of the fatty acids of DT-1 is typical of the B. cereus group. The profile of DT-2 is typical of the RNA group I of the pseudomonads, and the profile of DT-5 points to the Rhizobium group.
    Pseudomonas azelaica DT-6 is a 0.5 - 0.7 µm wide and 1.5 - 3.0 µm long gram-negative motile rod having 1 - 3 polar flagella and lacking fluorescent pigments. Its fatty acid analysis print (Figure 5) is typical of the RNA group I of the pseudomonads. The partial sequencing of the 16S rDNA shows a 99.8% similarity to Ps. azelaica. DT-6 has the following physiological reactions:
    Lysis by 3% KOH +
    Aminopeptidase (Cerny) +
    Oxidase +
    Catalase +
    ADH +
    NO2 from NO3 +
    Denitrification weak
    Urease -
    Hydrolysis of gelatin -
    Lecithinase -
    Utilization of (API 20NE)
    glucose +
    arabinose -
    adipat +
    malat +
    mannitol -
    gluconat +
    caprat +
    Azospirillum sp. DT-10 is a 0.8 - 1.2 µm wide and 2.0 - 4.0 µm long gram-negative rod. Its fatty acid analyisis print (Figure 6) is typical of the α-subgroup of the proteobacteria and points to the genus Azospirillum. The partial sequencing of the 16S rDNA shows similarities between 92% and 97.4% to different members of the genus Azospirillum. The highest similarity 97.4% was found to Azospirillum lipoferum. The physiological reactions of DT-10 are shown below. They point to the genus Azospirillum but are not typical of A. lipoferum. DT-10 is possibly a new species of this genus.
    Lysis by 3% KOH weak
    Aminopeptidase (Cerny) +
    Oxidase +
    Catalase +
    NO2 from NO3 +
    Urease +
    ADH -
    Hydrolysis of
    gelatin -
    esculin -
    Utilization of (sole carbon source) glucose -
    arabinose -
    adipat -
    malat +
    mannitol -
    phelyacetat -
    citrate -
    caprat -
    gluconat -
    maltose -
    n-acetylglucosamin -
    α-ketoglutarate +
    sucrose -
    m-inositol -
    D-fructose +
    rhamnose -
    arabitol -
    ribose -
    Growth at 41 °C -
    with 3% NaCl -
    Ancylobacter aquaticus DT-12 is a gram-negative curved rod which is 0.5 - 0.7 µm in width and 1.5 - 2.0 µm in length. The partial sequence of the 16S rDNA shows a similarity of 98.8% to Ancylobacter aquaticus. Thiobacillus novellus shows a similarity of 97.8%. The fatty acids (Figure 7) point to the α-proteobacteria. The physiological tests as shown below clearly identify the species Ancylobacter aquaticus.
    Lysis by 3% KOH weak
    Aminopeptidase (Cerny) +
    Oxidase +
    Catalase +
    ADH -
    Urease -
    Hydrolysis of gelatin -
    esculin +
    NO2 from NO3 -
    Denitrification (24 h) -
    Utilization of glucose + (weak)
    citrate +
    arabinose +
    mannose -
    mannitol +
    maltose -
    N-acetylglucosmin -
    gluconat -
    malat +
    phenylacetat -
    methanol +
    formiate weak
    Xanthobacter sp. DT-13 is an irregular, motile, gram-negative rod which is 0.8 - 1.0 µm in width and 1.5 - 3.0 µm in length. The partial sequences of the 16S rDNA show similarities of 98.5% to 99.3% to different members of the genus Xanthobacter. X. falvus shows the highest similarity (99.3%). The profile of the fatty acids is typical of the subclass of α-proteobacteria. The physiological tests are not able to distinguish reliably between the species of this genus (i.e. no pigment production detected, no slime production, etc.). The physiological data are given below:
    Lysis by 3% KOH +
    Aminopeptidase (Cerny) +
    Oxidase +
    Catalase +
    ADH -
    Urease (24 h) -
    Hydrolysis of gelatin -
    esculine -
    NO3 utilization -
    Utilization of phenylacetate -
    citrate -
    malate +
    arabinose -
    mannose -
    mannit -
    caprat -
    maltose -
    adipate +
    malonate +
    methanol -
    m-inosit -
    m-tartrate +
    D-gluconate +
    phelylalanine -
    The above-described bacteria are suited for use in purifying waste water. The bacteria can then be first grown in a minimal salt medium (KSN) in a shaker. Soy pepton (0.5 g/l), trypton (0.1 g/l), glucose (0.2 g/l) and potassium acetate (0.3 g/l) may be added, if desired. The growing temperature of the bacteria is about 20 - 30°C. After this, the volume of the culture is then increased in order to produce the necessary biomass for purifying the water. This stage no longer needs to be conducted under sterile conditions, in which case tap water wherein about 0.5 - 4 g/l of soap has been added can be used as the growth medium. The soap used is preferably a mixture containing anionic, cationic, amphoteric and non-ionic tensides. It is preferable to use a mixture of different soaps, such as cleaning agents, fabric conditioners and detergents for clothes and dishes. The bacteria are grown as a submerged culture with air pumped thereto. The biomass can be produced as a batch culture, but preferably, it is produced as a continuous culture, or chemostat culture. It is preferable to use a carrier in the production of the biomass. Any common carrier, for example a plastic one, is suitable for this purpose. The produced biomass is then transferred into a water treatment reactor, into which the water to be purified is conveyed. A carrier for the bacteria is also used in the reactor, the carrier preferably being the same as used in the production of the biomass. The carrier is preferably one having a specific density lower than 1 g/cm3. The carrier is generally held in place in a tank by means of a net ('fixed carrier'), for example, but sometimes the carrier is allowed to float freely in the tank ('swimming carrier').
    The method of the invention is suited particularly to purifying seep water of a dump, which is here described in closer detail with reference to Figure 1. A dump is usually surrounded by a ditch to collect the seep water. Seep water refers to water seeping from a dump due to rain and ground water. This seep water containing both surface water and cavity water is usually first conveyed to a tank wherefrom the water is conveyed through a purification process before being discharged into the environment. The seep water obtained both from deep and shallow ground is preferably first conveyed to a settlement basin, from which the water is filtered through an inlet pipe 1 to a filtrate well 2, and from there, through a transfer pipe 8 to a bioreactor 3 containing said bacteria and a carrier 5. The bacteria form a so-called biofilm around the carrier. The carrier with its bacteria is usually kept below the surface of the water by means of a net. The bioreactor preferably comprises one or more separating walls 6 arranged to force the water to circulate in the reactor. The separating walls may be arranged on opposite walls, for example, as shown in Figure 1. The reactor usually further comprises an aerator 9 for conveying air into the reactor through an aeration pipe 4. The bioreactor further comprises an outlet pipe 7, through which processed water is discharged from the reactor.
    In addition to purifying seep water, the present invention is extremely well suited also to purifying household and industrial grey water. Grey water refers to waste water other than that originating from lavatories, e.g. water from showers, handbasins, bath tubs and laundry rooms. The purification method of the invention is also suited to purifying waste water from lavatories, which is called black water. The method of the invention can also be used to purify laundry and industrial waste water, which often contains a large amount of organic waste, such as oil, polycyclic aromatic hydrocarbons (PAH compounds) and/or heavy metals. The method is also suitable for purifying waste water originating from food industry and water in swimming pools.
    Example 1 Production of biomass and start of a bioreactor
    Bacillus sp. DT-1, Pseudomonas azelaica DT-2 and Rhizobium sp. DT-5 were each transferred to 200 ml of sterilized minimum salt medium (KSN) of the following composition (g/l of distilled water): K2HPO4×3H2O - 1.0, NaH2PO4×2H2O - 0.25, (NH4)2SO4 - 0.1, MgSO4×7H2O - 0.04, Ca(NO3)2×4H2O - 0.01, yeast extract - 0.05, pH 7.0 - 7.3, and soap mixture about 1 g/l. The soap mixture contained about equal amounts of the following detergents: laundry soap, Comfort, Cleani Family -fabric conditioner, Cleani Color, Serto Ultra, Bio Luvil, Ariel Futur, Omo Color, Tend Color, Tend Mega, Tend Total and Eko Kompakt (about 1g/l in total). The bacteria were grown in a shaker (150 - 200 rpm), at 28°C.
    When the growth was dense, all three cultures were brought to one 500-litre fermenter in order to produce the necessary biomass. The fermenter contained unsterilized tap water and a total of 4 g/l of the aforementioned soap mixture, and a plastic carrier containing polyethene and having a specific density of about 0.8 g/cm3. The carrier was kept below the surface of the liquid by means of a net. The cultivation now continued under non-sterile conditions to a turbidity of about 2 (600 nm), and then as a chemostat culture. A first inoculum obtained from the fermenter was then introduced into a bioreactor (6 m3) according to Figure 1, diluted 1:10. The bioreactor contained seep water from a municipal dump which was first collected into a tank, wherefrom it was then transferred to a settlement basin for removal of solid matter and next, to a filtrate well, wherefrom it was pumped to the bioreactor. In principle, the system works by gravity, the only necessary pump being a submersible pump in the filtrate well. The bioreactor contained the same carrier as the fermenter used for producing the biomass. The carrier was kept below the liquid level by means of a net. The bacteria flocculated at the end of the bioreactor. The purification process was continuous, operating at a capacity of about 100 m3/24 hours. Air was pumped so as to keep the oxygen content of the water to be processed > 7 mg/l.
    Example 2 Purification of seep water
    A bioreactor arranged according to Example 1 was used for purification of seep water from a municipal dump. The average COD of the waste water to be purified was about 800 mg - 6 g O2/l. The waste water contained chlorophenoles, PAH compounds and oil, for example. The removal of these subsctances from the waste water was monitored. According to Nordtest's technical report no. 329 (accepted 9603), the compounds were defined by a gas chromatograph equipped with a mass-selective detector. The results are shown in Table 2.
    Detection Before bioreactor After bioreactor
    COD 0.8 - 6 g/l 100 - 200 mg/l
    chlorophenoles > 1 mg/l < 1 µg/l
    PAH
    1 mg/l < 1 µg/l
    oil 0.2 - 1 mg/l 200 µg/l
    Example 3 Purification of municipal waste water (full scale)
    Waste water from a municipal waste water plant was purified both in a manner conventionally used in the plant and by the method of the invention. Conventionally, waste water was purified by first conveying the waste water into a preliminary settlement basin in order to precipitate the solids onto the bottom. The preliminary settled water was then conveyed to an aerobic treatment basin, whereto ferrous sulphate for precipitating phosphate, and polyamine for precipitating biosludge were added. Herefrom, the water was further conveyed to a secondary settlement basin. The purification system of the invention comprised five tanks whose total volume was 7.5 m3, the tanks being interconnected in the following order: two anaerobic tanks, whereto bacteria DT-1, DT-2 and DT-5 were added without a carrier, one aerobic tank whereto a carrier was attached (by means of a net) on which the bacteria DT-1, DT-2 and DT-5 were immobilized, and two sedimentation tanks. The temperature was 8 - 15°C. The flow rate was 7.5 m3/24 hours of waste water. The aeration was conducted by recycling the water through the carrier. The results are shown in Table 3.
    Parameter Before treatment After conventional purification After purification of the invention
    BOD7 mg O2/l 200 - 300 10 - 15 10 - 15
    CODCr mg O2/l 250 - 500 60 - 75 40 - 50
    Total nitrogen mg N/l 35 - 55 15 - 25 15 - 25
    Total phosphor mg P/l 5 - 10 0.6 - 1.8 0.5 - 1.8
    Fec. streptococci cfu/100 ml 108 2 x 104- 3 x 104 2 x 104- 3 x 104
    Thermo-tolerant coliforms cfu/100 ml 3 x 108 2 x 104- 4 x 104 2 x 104- 4 x 104
    The purification results achieved by the method of the invention were either as good as or better than those achieved by the conventional method, and energy consumption was significantly lower. The energy consumption in treating one cubic metre of water was 0.23 kWh at the municipal waste water treatment plant, and 0.05 - 0.1 kWh when the method of the invention was used.
    Example 4 Purification of household black water (full scale)
    The system comprised five tanks whose total volume was 6.5 m3, the tanks being interconnected in the following order: two anaerobic tanks without a carrier into which the DT-1, DT-2 and DT-5 were added, one aerobic tank whereto a carrier was attached on which the bacteria DT-1, DT-2 and DT-5 were immobilized, and two sedimentation tanks. The temperature was 8 - 15°C. The flow rate was 0.5 - 5 m3 of waste water per 24 hours. The aeration was conducted by recycling the water through the carrier. The energy consumption was 0.05 - 0.5 kWh. The results are shown in Table 4.
    Parameter Before treatment After treatment
    BOD7 mg O2/l 400 - 5500 3 - 20
    CODcr mg O2/l 400 - 6000 40 - 70
    Total nitrogen mg N/l 100 - 300 1 - 5
    Total phosphorus mg P/l 10 - 25 0.2 - 2
    Fec. streptococci cfu/100 ml 108 -109 < 20
    Thermo-tolerant coliforms cfu/100 ml 108 - 109 < 20
    pH 7 - 8 6.5-7
    Example 5 Purification of industrial waste water containing soap and heavy metals (laboratory scale)
    Waste water from a coating metal industry plant was purified by a system whose effective treatment part comprised six anaerobic and twelve aerobic tanks. The bacteria DT-1, DT-2 and DT-5, which were immobilized on a carrier attached by nets, were added to all anaerobic and aerobic tanks. Each tank held 2 l. The entire system comprised 23 tanks whose total volume was 70 l, the tanks being interconnected in the following order: six anaerobic tanks (effective treatment volume), one sedimentation tank, six aerobic tanks (effective treatment volume), one sedimentation tank, six aerobic tanks (effective treatment volume), and two tanks for calcium chloride and sodium hydroxide treatments to precipitate the biomass and heavy metals. Before the treatment, the original waste water was diluted five times by gray water. After the dilution, mineral salts were added as follows: NH + / 4 2 - 10 mg/l, NO - / 3 5 - 20 mg/l. Mg2+ 2 - 10 mg/l, Ca2+ 0.5 - 2 mg/l, SO4 2- 1 - 10 mg/l and PO4 3- 2 - 20. mg/l. The temperature was 20 - 35°C and the flow rate 12 l of water per 24 hours. The results are shown in Table 5.
    Parameter Before treatment After treatment
    CODCr mg O2/l 19 000 - 21 000 100 - 400
    Total phosphorus mg P/I 19 - 25 0.3 - 0.7
    Aluminium 5 - 6 0.01 - 0.02
    Chrome 1.3 - 1.5 0.01 - 0.02
    Copper 35 - 40 0.03 - 0.1
    Iron 1 - 2 0.02 - 0.07
    Lead 23 - 25 0.02 - 0.09
    Nickel 2 - 3 0.05 - 0.09
    Zinc 30 - 60 0.003 - 0.007
    pH 8-9 7 - 7.5
    Example 6 Purification of household grey water for recycling (pilot scale)
    The effective part of the system comprised three aerobic tanks whose single volume was 0.2 m3. The entire system comprised six tanks whose total volume was 2.8 m3, the tanks being interconnected in the following order: one tank for collecting grey water, three aerobic tanks comprising a fixed carrier on which the bacteria DT-1, DT-2 and DT-5 were immobilized (effective treatment volume), one aerobic tank without a carrier and one sedimentation tank, and, subsequently, a filtering system and a UV-light treatment system. The temperature was 20 - 35°C. The flow rate was about 1 m3 per 24 hours. The results are shown in Table 6.
    Parameter Before treatment After treatment
    CODCr mg O2/l 150-400 15 - 35
    Total nitrogen mg N/l 10-15 < 0.5
    Total phosphorus mg P/l 5 -10 < 0.1
    Coliforms cfu/100 ml 1.4 - 2 x 106 0
    pH 7.5-8.5 6.5-7
    Example 7 Purification of grey water of a laundry for recycling (pilot scale)
    The effective treatment part of the system comprised two aerobic tanks having the volume of 1 m3, the tanks comprising a swimming carrier on which DT-1, DT-2 and DT-5 were immobilized. The entire system comprised ten tanks whose total volume was 23 m3, the tanks being interconnected in the following order: one tank for collecting grey water, two aerobic tanks comprising a swimming carrier (effective treatment volume), one sedimentation tank, three aerobic tanks comprising a fixed carrier with its bacteria (effective treatment volume), one aerobic tank without a carrier, and two sedimentation tanks. The temperature of the water was 20 - 35°C, the flow rate 1 m3 of waste water per 24 hours. The results are shown in Table 7.
    Parameter Before treatment After treatment
    CODcr mg O2/l 200 - 450 25 - 35
    Total phosphorus mg P/l 1 - 2 < 0.1
    pH 8.5-9 7-8
    Example 8 Increase of immobilized biomass
    Biomass of strains DT-1, DT-2, DT-5, DT-6, DT-10, DT-12 and DT-13 was produced and immobilzed on a carrier as set forth in Example 1, and the amount of biomass on the carrier was weighed. The weight of one disc of the carrier was 72 ± 1 g. When DT-1, DT-2 and DT-5 were immobilized on the carrier, the weight of one disc of the carrier was 119 ± 13, i.e. the wet weight of the biomass was 47 ± 11 g per disc. When all seven bacterial strains were immobilized on the carrier, the weight of one disc of carrier was 172 ± 16, i.e. the wet weight of the biomass was 91 ± 16. The results show that DT-6, DT-10, DT-12 and DT-13 increased the immobilized biomass about twofold.

    Claims (19)

    1. A method of purifying waste water, characterized in that the water is biologically purified by a mixed population comprising the microorganisms Bacillus sp. DT-1 having the deposit number DSM 12560. Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and Rhizobium sp. DT-5 having the deposit number DSM 12562.
    2. A method as claimed in claim 1, characterized by purifying seep water, grey water, black water, industrial waste water and waste water from laundries.
    3. A method as claimed in claim 1 or 2, characterized in that necessary biomass for the purification is produced in a non-sterilized growth medium comprising tap water and about 0.5 - 4 g/l of soap.
    4. A method as claimed in claim 1, characterized in that the water is also purified by one or more microorganisms from the group Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and Xanthobacter sp. DT-13 having the deposit number DSM 13519.
    5. Bacillus sp. DT-1 having the deposit number DSM 12560.
    6. Pseudomonas azelaica DT-2 having the deposit number DSM 12561.
    7. Rhizobium sp. DT-5 having the deposit number DSM 12562.
    8. Pseudomonas azelaica DT-6 having the deposit number DSM 13516.
    9. Azospirillium sp. DT-10 having the deposit number DSM 13517.
    10. Ancylobacter aquaticus DT-12 having the deposit number DSM 13518.
    11. Xanthobacter sp. DT-13 having the deposit number DSM 13519.
    12. A bacterial mixed population, characterized by comprising Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and/or Rhizobium sp. DT-5 having the deposit number DSM 12562.
    13. A bacterial mixed population as claimed in claim 12, characterized by further comprising Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and/or Xanthobacter sp. DT-13 having the deposit number DSM 13519.
    14. Use of a bacterial mixed population as claimed in claim 1 or 4 in purifying waste water.
    15. A bioreactor, characterized by comprising the microorganisms Bacillus sp. DT-1 having the deposit number DSM 12560, Pseudomonas azelaica DT-2 having the deposit number DSM 12561, and Rhizobium sp. DT-5 having the deposit number DSM 12562.
    16. A bioreactor as claimed in claim 15, characterized by further comprising one or more microorganisms from the group Pseudomonas azelaica DT-6 having the deposit number DSM 13516, Azospirillium sp. DT-10 having the deposit number DSM 13517, Ancylobacter aquaticus DT-12 having the deposit number DSM 13518, and Xanthobacter sp. DT-13 having the deposit number DSM 13519.
    17. A bioreactor as claimed in claim 16, characterized by comprising all said seven bacterial strains.
    18. A bioreactor as claimed in claim 15, characterized by comprising one or more separating walls arranged so as to force water to circulate in the reactor.
    19. A bioreactor as claimed in claim 18, characterized in that the bacteria are immobilized on a plastic carrier medium whose specific density is about 0.8 g/cm3,
    EP00948037A 1999-07-12 2000-07-06 Method of purifying water, suitable bacteria for the method and use thereof Expired - Lifetime EP1204608B9 (en)

    Applications Claiming Priority (3)

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    FI991595 1999-07-12
    FI991595 1999-07-12
    PCT/FI2000/000624 WO2001004060A2 (en) 1999-07-12 2000-07-06 Method of purifying water, suitable bacteria for the method and use thereof

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    CA2357907A1 (en) 2001-09-26 2003-03-26 Garfield R. Lord Bacteria growth apparatus for use in multi chamber biological reactor used after a settling tank or solids removal apparatus
    KR100437103B1 (en) * 2002-02-08 2004-06-23 최동성 Pseudomonas sp. FK 916 producing decomposition enzyme of organic compound and method for decomposition of organic waste using the same
    EP2216398A3 (en) * 2005-04-21 2010-10-27 Ibiden Co., Ltd. Method of treating wastewater containing organic compound
    DE102005025562A1 (en) * 2005-06-01 2006-12-14 Syntana Gmbh Denitrification of activated sludge comprises addition of raw glycerin as external source of carbon to a biological sewage purification plant for reducing phosphorus content during last quarter of biological phosphorus elimination process
    GB201003200D0 (en) 2010-02-25 2010-04-14 Microbial Solutions Ltd Method for the treatment of industrial waste
    CN103013858A (en) * 2012-11-21 2013-04-03 浙江工业大学 Ancylobacter Tet-1 and application thereof in microbial degradation of tetramethrin
    US20140154784A1 (en) * 2012-12-03 2014-06-05 Gregory van Buskirk Method and Process for the Degradation of Cyclic Ethers in Ethoxylate-Containing Actives
    CN103241843B (en) * 2013-05-24 2015-12-02 安徽美自然环境科技有限公司 The Thermal insulation biological medium oxidation treatment method of sanitary sewage
    GB2545875B (en) * 2015-07-30 2018-06-06 Ford Motor Co Consortium
    CN105668807A (en) * 2016-03-01 2016-06-15 云南圣清环保科技有限公司 Method adopting microorganisms for treating coal chemical industrial wastewater
    FI127756B (en) 2017-04-24 2019-02-15 Clewer Aquaculture Oy Bioreactor

    Family Cites Families (7)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    US4317885A (en) 1979-05-01 1982-03-02 Sybron Corporation Microbiological process for removing non-ionic surface active agents, detergents and the like from wastewater and microorganism capable of same
    GB8701555D0 (en) 1987-01-24 1987-02-25 Kambanellas C A Liquid filtration
    US5245711A (en) 1988-09-06 1993-09-21 Oy Wartsila Ab Vacuum toilet system
    WO1994005866A1 (en) 1992-09-03 1994-03-17 Hydrosave Recycling Systems Pty Ltd Apparatus and method for waste water recycling
    DE69516637T2 (en) 1994-11-21 2000-09-21 Canon Kk Process for the degradation of pollutants and environmental remediation using microorganisms and the microorganism used
    EP0915061A1 (en) 1997-10-20 1999-05-12 Realco 2001 S.A./N.V. Method for reducing the effect of detergents upon germination and/or growth of micro-organisms
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    MXPA02000504A (en) 2004-05-21
    EP1204608A2 (en) 2002-05-15
    WO2001004060A2 (en) 2001-01-18
    DE60017263T2 (en) 2006-01-26
    WO2001004060A3 (en) 2001-07-19
    US6780317B1 (en) 2004-08-24
    PL352611A1 (en) 2003-08-25
    CN1420848A (en) 2003-05-28
    JP2003504185A (en) 2003-02-04
    HUP0202345A2 (en) 2002-10-28
    EP1204608B1 (en) 2005-01-05
    CN1183046C (en) 2005-01-05
    ATE286487T1 (en) 2005-01-15

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